Antioxidants can be described as compounds (e.g., enzymes, organic molecules) that slow the rate of oxidation reactions or that can counteract the damaging effects of oxygen. Although the term technically applies to molecules reacting with oxygen, it is often applied to molecules that protect from any free radical (i.e., a molecule with an unpaired electron, such as hydroxyl radicals, lipid oxyl or peroxyl radicals, singlet oxygen, and peroxinitrite formed from nitrogen oxide). Free radicals are natural by-products of cellular processes in an organism or are created by exposure to environmental factors. Within cellular organisms, free radicals can cause cellular and tissue damage, which can ultimately lead to disease. Antioxidants neutralize free radicals by donating one of their own electrons to the free radical, since the radicalized antioxidant molecule is more stable as a free-radical than the original free-radical.
A variety of nutrients or dietary components have antioxidant properties and thus can function to decrease the tissue content of reactive oxygen. Common antioxidants include vitamins C and E, β-carotene, proanthocyanidin, the minerals selenium and zinc, and coenzyme Q. Coenzyme Q, also known as ubiquinone and referred to herein as “CoQ”, refers to a series of related 2-3-dimethoxy-5-methyl-benzoquinones with a polyisoprenoid side chain in the 6-position that are widely distributed in animals, plants and microorganisms.
In structure, the CoQ group closely resembles the members of the vitamin K group and the tocopherylquinones (derived from tocopherols, e.g., vitamin E) in that they all possess: 1) a quinonoid ring derived from tyrosine or phenylalanine that functions as an electron-carrier; and, 2) a long hydrocarbon tail comprised of 5-carbon isoprene units. The quinones of the CoQ series, that is Q6, Q7, Q8, Q9 and Q10, found in various biological species differ only slightly in chemical structure based on the length of the hydrocarbon tail, which ranges from 30 to 50 carbon atoms (corresponding to 6, 7, 8, 9 or 10 isoprenoid units in the side chain) and which facilitates CoQ's localization in mitochondrial or cytoplasmic membranes. Differences in properties are due to the differences in length of the side chain.
The antioxidant properties of CoQ10 are directly related to the coenzyme's bioenergetic functions. Specifically, CoQ10 is involved in the terminal electron transport system by transporting electrons from organic substrates to oxygen in the respiratory chain of mitochondria, which is essential in the production of biochemical energy (e.g., ATP) in all cells of aerobic organisms. As an energy carrier, CoQ10 is continually going through an oxidation-reduction cycle. Specifically, CoQ10 is reduced to a free radical semiquinone by the uptake of a single electron; reduction of this enzyme-bound intermediate by a second electron yields ubiquinol. As ubiquinol, the molecule holds electrons loosely and can easily donate one or two electrons to neutralize free radicals, which can be created in part by the energy-generating process.
The antioxidant properties of CoQ10 are significant. In addition to quenching free radicals that threaten cellular components, such as nucleic acids and proteins in the mitochondria, ubiquinol also inhibits lipid peroxidation (i.e., degradation of lipids) in biological membranes and in low-density lipoprotein [“LDL”]. Furthermore, functionality of CoQ10 may be enhanced in the presence of carotenoid compounds (see Int'l. App. Pub. No. WO 2005/097091 A1).
Based on the physiological role that CoQ10 plays within living organisms, the coenzyme has become widely used as a nutritional supplement and as a pharmacological active agent. It has wide use and acceptance in the treatment of: mitochondrial disorders, cardiovascular disease processes, atherosclerosis, slow muscle degeneration (dystrophy or atrophy), neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease, Alzheimer's, amyotrophic lateral sclerosis [“ALS”]), periodontal disease, diabetes and CoQ10 deficiency. CoQ10 is also believed to strengthen the immune system, act as an anticancer agent and help counteract the aging processes.
CoQ10 is currently available via chemical synthesis, semi-chemical synthesis and microbial conversion (Choi, Jin-Ho et al., Appl. Microbiol. Biotechnol., 68:9-15 (2005)). In the biotechnological arena, several strains of Agrobacterium tumefaciens, A. radiobacter, Rhodobacter sphaeroides and Paracoccus denitrificans have been identified that produce CoQ10 in significant quantities (Yoshida et al., J. Gen. Appl. Microbiol., 44:19-26 (1998)), and marine bacteria of the genus Erythrobacter, Sphingomonas, Exiguobacterium, Lutibacterium and Bacillus have also been found to naturally produce CoQ10 (Int'l. App. Pub. No. WO 2008023264). Genetic engineering of microbes, such as Escherichia coli, Rhodobacter sphaeroides, and plants such as brown rice for CoQ10 biosynthesis has also been demonstrated with the expression of heterologous genes encoding decaprenyl diphosphate synthase (e.g., Zahiri et al., Metabol. Engineering, 8:406-416 (2006); JP 10057072; JP 2005211020; JP 2006 204215; Int'l. App. Pub. No. WO 00/047746; Int'l. App. Pub. No. WO 02/026933; U.S. Pat. No. 6,461,842; U.S. Pat. App. Pub. No. 2006/010519; Int'l. App. Pub. No. WO 07/120423). An oleaginous microbial host cell having the ability to co-produce CoQ10 and at least 25% of its dry cell weight [“DCW”] as oil, wherein the CoQ10 can advantageously help protect against autoxidation of the oil is expected to be advantageous. Both CoQ10 and oil can be extracted with hexane or other solvents, thus reducing production cost. The offering of a final product containing both ingredients, that is, a stabilized microbial oil, may command a higher premium or competitive advantage.
An oleaginous microbial host cell that can co-produce CoQ10 and polyunsaturated fatty acids [“PUFAs”] has not been reported. This deficiency exists despite previous descriptions of the utility of co-administration of CoQ10 with PUFAs (see e.g., U.S. Pat. App. Pub. No. 2002/0198177 A1) and despite previous recognition that identification of such a microbe would be advantageous (e.g., see screening studies of various Thraustochytrids (marine fungoid protists) by Ocean Nutrition Canada, Ltd., as described in Burja et al., Appl. Microbiol. Biotechnol., 72:1161-1169 (2006) and Armenta et al., J. Agric, Food Chem., 54:9752-9758 (2006)). A means to recombinantly produce both CoQ10 and PUFAs in a single microbial host cell would create a single product comprising both ingredients. This is particularly attractive when the recombinant cell biomass is used directly in the formulation, such as an animal feed.
Additionally, there are no reports of a microbial host cell that can co-produce CoQ10, PUFAs and carotenoids, wherein said host cell comprises at least 25% of its DCW as oil, although it is recognized that the functionality of CoQ10 may be enhanced in the presence of carotenoid compounds (see Int'l. App. Pub. No. WO 2005/097091 A1).
Carotenoids are themselves generally classified as antioxidants and may help to protect one another from oxidation during production and/or storage. As such, some carotenoids may alternatively be viewed as natural antioxidants in certain product applications where the carotenoid is not used as a pigment; for example, use of lycopene as an antioxidant in food products and/or animal feeds.
Many commercial products are formulated to contain a mixture of natural antioxidants, such as CoQ10, and fats/lipids and/or pigments. For example, animal feeds, dietary supplements, and personal care products are formulated to contain antioxidants, PUFAs and carotenoids. Typically, for example, a commercial product formulator will obtain these compounds from a variety of sources and formulate them into a final product that contains an effective amount of each ingredient. The composition, purity and source of each ingredient may vary, resulting in a final product formulation that may require significant monitoring and/or processing to obtain the desired product specifications.
Engineering an oleaginous microorganism to simultaneously produce both CoQ10 and at least one ω-3/ω-6 PUFA (and optionally at least one C40 carotenoid) would create a higher value product or reduce production costs. Since carotenoids and PUFAs may undergo oxidation during storage, materials comprising these compounds are typically supplemented with one or more antioxidants. However, many of the synthetic antioxidants currently used in the market are undesirable due to their cost and/or possible safety concerns. If a microbial host produces both carotenoids and PUFAs in conjunction with a reduced form of CoQ10, the CoQ10 may aid in protecting compositions comprising carotenoids, PUFAs, and mixtures thereof from oxidation.
The problem to be solved therefore, is to provide a recombinant oleaginous yeast capable of producing the antioxidant CoQ10 in combination with at least one ω-3/ω-6 PUFA (and optionally at least one C40 carotenoid).